Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Arabidopsis cytosolic alpha‐glycan phosphorylase,PHS2, is important during carbohydrate imbalanced conditions

Arabidopsis thaliana has two isoforms of alpha‐glycan phosphorylase (EC 2.4.1.1), one residing in the plastid and the other in the cytosol. The cytosolic phosphorylase, PHS2, acts on soluble heteroglycans that constitute a part of the carbohydrate pool in a plant. This study aimed to define a physiological role for PHS2. Under standard growth conditions phs2 knock‐out mutants do not show any clear growth phenotype, and we hypothesised that during low‐light conditions where carbohydrate imbalance is perturbed, this enzyme is important. Soil‐grown phs2 mutant plants developed leaf lesions when placed in very low light. Analysis of soluble heteroglycan (SHG) levels showed that the amount of glucose residues in SHG was higher in the phs2 mutant compared to wild‐type plants. Furthermore, a standard senescence assay from soil‐grown phs2 mutant plants showed that leaves senesced significantly faster in darkness than the wild‐type leaves. We also found decreased hypocotyl extension in in vitro‐grown phs2 mutant seedlings when grown for long time in darkness at 6 °C. We conclude that PHS2 activity is important in the adult stage during low‐light conditions and senescence, as well as during prolonged seedling development when carbohydrate levels are unbalanced.     

Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana

Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.     

Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells

In plants several ‘starch-related’ enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.     

A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)

The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step ¹⁴C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- α -arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.     

Alterations in cytosolic glucose-phosphate metabolism affect structural features and biochemical properties of starch-related heteroglycans

The cytosolic pools of glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate are essential intermediates in several biosynthetic paths, including the formation of sucrose and cell wall constituents, and they are also linked to the cytosolic starch-related heteroglycans. In this work, structural features and biochemical properties of starch-related heteroglycans were analyzed as affected by the cytosolic glucose monophosphate metabolism using both source and sink organs from wild-type and various transgenic potato (Solanum tuberosum) plants. In leaves, increased levels of the cytosolic phosphoglucomutase (cPGM) did affect the cytosolic heteroglycans, as both the glucosyl content and the size distribution were diminished. By contrast, underexpression of cPGM resulted in an unchanged size distribution and an unaltered or even increased glucosyl content of the heteroglycans. Heteroglycans prepared from potato tubers were found to be similar to those from leaves but were not significantly affected by the level of cPGM activity. However, external glucose or Glc-1-P exerted entirely different effects on the cytosolic heteroglycans when added to tuber discs. Glucose was directed mainly toward starch and cell wall material, but incorporation into the constituents of the cytosolic heteroglycans was very low and roughly reflected the relative monomeric abundance. By contrast, Glc-1-P was selectively taken up by the tuber discs and resulted in a fast increase in the glucosyl content of the heteroglycans that quantitatively reflected the level of the cytosolic phosphorylase activity. Based on (14)C labeling experiments, we propose that in the cytosol, glucose and Glc-1-P are metabolized by largely separated paths.     

A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Aphid-repellent pheromone E-β-farnesene is generated in transgenic Arabidopsis thaliana over-expressing farnesyl diphosphate synthase2

Background and Aims Plant-synthesized sesquiterpenes play a pivotal role in chemotactic interactions with insects. Biosynthesis of functionally diverse sesquiterpenes is dependent on the availability of a pool of the precursor farnesyldiphosphate (FDP). In Arabidopsis thaliana, FPS2, encoding cytosolic farnesyldiphosphate synthase, is implicated in the synthesis of cytosolic FDP, but it is not known whether enhanced levels of FDP have a commensurate effect on sesquiterpene-mediated defence responses. This study examined transgenic arabidopsis plants generated to over-express FPS2 in order to determine if any effects could be observed in the response of aphids, Myzus persicae.Methods Transgenic arabidopsis plants were generated to over-express FPS2 to produce FPS2 in either the cytosol or the chloroplasts. Morphochemical analyses of the transgenic plants were carried out to detremine growth responses of roots and shoots, and for GC-MS profiling of sesquiterpenes. Aphid response to hyrdo-distillate extracts and head-space volatiles from transgenic plants was assessed using a bioassay.Key Results Either over-expression of FPS2 in the cytosol or targetting of its translated product to chlorplasts resulted in stimulatory growth responses of transgenic arabidopsis at early and late developmental stages. GC-MS analysis of hydro-distillate extracts from aerial parts of the plants revealed biosynthesis of several novel sesquiterpenes, including E-β-farnesene, an alarm pheromone of aphids. Both entrapped volatiles and hydro-distillate extracts of the transgenic leaves triggered agitation in aphids, which was related to both time and dose of exposure.Conclusions Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in arabidopsis led to synthesis of several novel sesquiterpenes, including E-β-farnesene, and induced alarm responses in M. persicae. The results suggest a potential for engineering aphid-resistant strains of arabidopsis.     

GPT2: a glucose 6-phosphate/phosphate translocator with a novel role in the regulation of sugar signalling during seedling development

Background and Aims

GPT2, a glucose 6-phosphate/phosphate translocator, plays an important role in environmental sensing in mature leaves of Arabidopsis thaliana. Its expression has also been detected in arabidopsis seeds and seedlings. In order to examine the role of this protein early in development, germination and seedling growth were studied.

Methods

Germination, greening and establishment of seedlings were monitored in both wild-type Arabidopsis thaliana and in a gpt2 T-DNA insertion knockout line. Seeds were sown on agar plates in the presence or absence of glucose and abscisic acid. Relative expression of GPT2 in seedlings was measured using quantitative PCR.

Key Results

Plants lacking GPT2 expression were delayed (25–40 %) in seedling establishment, specifically in the process of cotyledon greening (rather than germination). This phenotype could not be rescued by glucose in the growth medium, with greening being hypersensitive to glucose. Germination itself was, however, hyposensitive to glucose in the gpt2 mutant.

Conclusions

The expression of GPT2 modulates seedling development and plays a crucial role in determining the response of seedlings to exogenous sugars during their establishment. This allows us to conclude that endogenous sugar signals function in controlling germination and the transition from heterotrophic to autotrophic growth, and that the partitioning of glucose 6-phosphate, or related metabolites, between the cytosol and the plastid modulates these developmental responses.     

Measuring whole plant CO2 exchange with the environment reveals opposing effects of the gin2–1 mutation in shoots and roots of Arabidopsis thaliana

Using a cuvette for simultaneous measurement of net photosynthesis in above ground plant organs and root respiration we investigated the effect of reduced leaf glucokinase activity on plant carbon balance. The gin2–1 mutant of Arabidopsis thaliana is characterized by a 50% reduction of glucokinase activity in the shoot, while activity in roots is about fivefold higher and similar to wild type plants. High levels of sucrose accumulating in leaves during the light period correlated with elevated root respiration in gin2–1. Despite substantial respiratory losses in roots, growth retardation was moderate, probably because photosynthetic carbon fixation was simultaneously elevated in gin2–1. Our data indicate that futile cycling of sucrose in shoots exerts a reduction on net CO₂ gain, but this is over-compensated by the prevention of exaggerated root respiration resulting from high sucrose concentration in leaf tissue.     

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.     

Starch phosphorylase: Role in starch metabolism and biotechnological applications

The α-glucan phosphorylases of the glycosyltransferase family are important enzymes of carbohydrate metabolism in prokaryotes and eukaryotes. The plant α-glucan phosphorylase, commonly called starch phosphorylase (EC 2.4.1.1), is largely known for the phosphorolytic degradation of starch. Starch phosphorylase catalyzes the reversible transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of α-1,4-d-glucan chains with the release of phosphate. Two distinct forms of starch phosphorylase, plastidic phosphorylase and cytosolic phosphorylase, have been consistently observed in higher plants. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose-1-phosphate and for the development of engineered varieties of glucans and starch. Despite several investigations, the precise functional mechanisms of its characteristic multiple forms and the structural details are still eluding us. Recent discoveries have shed some light on their physiological substrates, precise biological functions, and regulatory aspects. In this review, we have highlighted important developments in understanding the role of starch phosphorylases and their emerging applications in industry.     

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

Background and Aims

Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa.

Methods

Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn²⁺. Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E_M) of mature cortical root cells and changes in the pH of the liquid culture media were measured.

Key Results

Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn²⁺ concentrations than A. thaliana plants. Elevated Zn²⁺ concentrations in the culture medium induced rapid changes in E_M. The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn²⁺-induced lower pH.

Conclusions

The results indicate a similar mode of early reaction to Zn²⁺, but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.     

Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.     

Neuropeptide Receptors NPR-1 and NPR-2 Regulate Caenorhabditis elegans Avoidance Response to the Plant Stress Hormone Methyl Salicylate

Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-messengerRNA (mRNA) splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen recovered Arabidopsis RTF2 (AtRTF2), a previously uncharacterized, evolutionarily conserved protein containing a replication termination factor 2 (Rtf2) domain. A homozygous null mutation in AtRTF2 is embryo lethal, indicating that AtRTF2 is an essential protein. Quantitative RT-PCR demonstrated that impaired expression of GFP in atrtf2 and prp8 mutants is due to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA sequencing indicated that 13–16% of total introns are retained to a significant degree in atrtf2 mutants. Considering these results and previous suggestions that Rtf2 represents an ubiquitin-related domain, we discuss the possible role of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing.     

Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.

Guard cells synthesize starch using carbon precursors originating in the plastid and/or imported from the cytosol depending on the time of the day.     

The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells

Transitory starch is stored during the day inside chloroplasts and then broken down at night for export. Recent data indicate that maltose is the major form of carbon exported from the chloroplast at night but its fate in the cytosol is unknown. An amylomaltase gene (malQ) cloned from Escherichia coli is necessary for maltose metabolism in E. coli. We investigated whether there is an amylomaltase in the cytosol of plant leaves and the role of this enzyme in plants. Two mutants of Arabidopsis thaliana (L) Heynh. were identified in which the gene encoding a putative amylomaltase enzyme [disproportionating enzyme 2, DPE2 (DPE1 refers to the plastid version of this enzyme)] was disrupted by a T-DNA insertion. Both dpe2-1 and dpe2-2 plants exhibited a dwarf phenotype and accumulated a large amount of maltose. In addition, dpe2 mutants accumulated starch and a water-soluble, ethanol/KCl-insoluble maltodextrin in their chloroplasts. At night, the amount of sucrose in dpe2 plants was lower than that in wild-type plants. These results show that Arabidopsis has an amylomaltase that is involved in the conversion of maltose to sucrose in the cytosol. We hypothesize that knocking out amylomaltase blocks the conversion from maltose to sucrose, and that the higher amount of maltose feeds back to limit starch degradation reactions in chloroplasts. As a result, dpe2 plants have higher maltose, higher starch, and higher maltodextrin but lower nighttime sucrose than wild-type plants. Finally, we propose that maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in the cytoplasm of E. coli.Abbreviations F6P fructose 6-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - GTase glucanotransferase     

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

Background and Aims

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process.

Methods

A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated.

Key Results

Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones.

Conclusions

No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.